1. Field of the Invention
The invention relates to cancer diagnosis and treatment, and specifically to the determination of an angiogenic phenotype of vascular endothelial cells from cancer patients. The invention specifically relates to the separation of vascular endothelial cells from non-endothelial cells, particularly tumor cells, in human tumor samples. The invention in particular relates to the identification of genes that are differentially expressed in anti-angiogenic agent-sensitive vascular endothelial cells compared with the expression of these genes in anti-angiogenic agent-resistant vascular endothelial cells, or compared to the expression of these genes in vascular endothelial cells that are not exposed to the drug. As part of this identification, the invention provides a pattern of expression from a selected number of identified genes, the expression of which is increased or decreased in anti-angiogenic agent-resistant vascular endothelial cells. The invention provides methods for identifying such genes and expression patterns of such genes and using this information to identify new gene targets for rational drug design, to identify new anti-angiogenic agents, and to make clinical decisions on cancer treatment, especially chemotherapeutic drug treatment of cancer patients.
2. Summary of the Related Art
Cancer remains one of the leading causes of death in the United States. Clinically, a broad variety of medical approaches, including surgery, radiation therapy and chemotherapeutic drug therapy are currently being used in the treatment of human cancer (see the textbook CANCER: Principles & Practice of Oncology, 6th Edition, De Vita et al., eds., J. B. Lippincott Company, Philadelphia, Pa.,2001). However, it is recognized that such approaches continue to be limited by a fundamental inability to accurately predict the likelihood of clinically successful outcome, particularly with regard to the sensitivity or resistance of a particular patient's tumor to a chemotherapeutic agent or combinations of chemotherapeutic agents.
A broad variety of chemotherapeutic agents are used in the treatment of human cancer. These include the plant alkaloids vincristine, vinblastine, vindesine, and VM-26; the antibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin, mitomycin C and bleomycin; the antimetabolites methotrexate, 5-fluorouracil, 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agents cyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU, streptozotocin, chlorambucil, bis-diamminedichloroplatinum, azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSA and mitoxantrone (Id., DeVita et al.).
However, some neoplastic cells become resistant to specific chemotherapeutic agents, in some instances even to multiple chemotherapeutic agents, and some tumors are intrinsically resistant to certain chemotherapeutic agents. Such drug resistance or multiple drug resistance can theoretically arise from expression of genes that confer resistance to the agent, or from lack of expression of genes that make the cells sensitive to a particular anticancer drug. One example of the former type is the multidrug resistance gene, MDR1, which encodes an integral plasma membrane protein termed P-glycoprotein that is a non-specific, energy-dependent efflux pump. (See Roninson (ed)., 1991, Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, N.Y., 1991; Gottesman et al., 1991, in Biochemical Bases for Multidrug Resistance in Cancer, Academic Press, N.Y., Chapter 11 for reviews). Examples of the latter type include topoisomerase II, the expression of which makes cells sensitive to the anticancer drug etoposide. Decreased expression of this enzyme makes neoplastic cells resistant to this drug. (See Gudkov et al., 1993, Proc. Natl. Acad. Sci. USA 90: 3231-3235). Although these are just single examples of the way that modulation of gene expression can influence chemotherapeutic drug sensitivity or resistance in neoplastic cells, these examples demonstrate the diagnostic and prognostic potential for identifying genes the expression of which (or the pattern of gene expression modulation thereof) are involved in mediating the clinical effectiveness of anticancer drug treatment.
Drug discovery programs have evolved to include rational therapeutic development strategies in addition to traditional empirical screening approaches. Rational therapy development focuses on the identification of specific pathways that are differentially activated in cancer cells compared to normal tissue (Bichsel et al., 2001, Cancer J. 7: 69-78; Winters, 2000, Curr. Opin. Mol. Ther. 2: 670-681). Such selective targeting can significantly reduce therapy-associated toxicity. Recent examples where this approach has led to the successful development of new anti-cancer agents include targeting HER2 with Herceptin (Bange et al., 2001, Nat. Med. 7: 548-552) in breast cancer and Gleevec's (STI571) inhibition of the BCR-ab1 kinase fusion protein in chronic myeloid leukemia (2001, Oncology (Huntingt) 15: 23-31).
Unfortunately, cancer specific pathways are not universal to the transformation process. Transformation results from a variety of alterations in tumor suppressor genes, oncogenes, translocations, deletions and mutations. The genomic instability inherent to this pleiotropic background of metabolic alterations results in significant phenotypic heterogeneity within each tumor (Bertram, 2000, Mol. Aspects Med. 21: 167-223; Yamasaki et al., 2000, Toxicol. Lett. 112-113: 251-256). Treatment targets are therefore unstable, leading to intrinsic and acquired resistance to rationally designed agents.
Angiogenesis, on the other hand, is a highly regulated process controlled by conserved gene cassettes (Folkman et al., 1992, J. Biol. Chem. 267: 10931-10934; Battery et al., 1995, J. Mol. Med. 73: 333-346). Recruitment of resting vascular endothelial cells (“VEC”) in response to the increased metabolic demands of a growing tumor mass follows stable pathways that are normally invoked in wound healing, reproductive physiology, and in ontogeny (Sen et al., 2002, Am. J. Physiol. Heart Circ. Physiol. 282: H1821-7) Thus, evaluation of these pathways offers a distinct advantage for rational therapeutic design because of their intrinsic stability (Schnitzer et al., 1998, New England J. Med. 339: 472-474; Jones et al., 2000, Principles and Practices of Oncology Updates 14:1-9).
Although mechanisms of angiogenesis in normal tissues have been extensively studied using traditional molecular biology, biochemical and immunological methods (reviewed in Saaristo et al., 2000, Oncogene 19: 6122-6129), the prior art contains sparse disclosure relating to differential gene expression in VECs. Li et al. (2001, J. Cereb. Blood Flow Metab. 21: 61-68) developed a protocol for purifying mRNA from isolated normal rat brain capillaries and subsequent microarray analysis of genes selectively expressed in the blood-brain barrier. They identified a series of over 40 novel gene sequences and known genes, including tissue plasminogen activator (TPA), insulin-like growth factor-2, regulators of G protein signaling, etc.), that had not been known to be specific for the blood-brain barrier functions. Similar experiments on normal bone marrow VEC using Atlas cDNA gene arrays showed the presence of mRNAs of several hematopoietic stimulators, cytokines and interleukins, in these cells (Li et al., 2000, Cytokine 12: 1017-1023). cDNA microarray analysis of 268 human VEC genes following infection with Chlamydia pneumoniae compared with uninfected endothelial cells revealed 20 genes up-regulated in response to C. pneumoniae infection, including cytokines (IL-1), chemokines (IL-8, monocyte chemotactic protein 1), and cellular growth factors, including basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) (Coombes et al., 2001, Infect. Immunol. 69: 1420-1427). Microarray-based evaluation of transcriptional profiles of mechanically induced genes in normal human aortic VEC using vascular endothelial growth factor (VEGF) as a positive control identified 3 out of 5000 transcripts up-regulated in these cells (cyclooxygenase-1, tenascin-C, and TPA-1; Feng et al., 1999, Circ. Res. 85:1118-1123). Down-regulated genes included thrombomodulin and matrix metalloproteinase-1 (MMP). Recently, Zhang et al. (Physiol. Genomics 5: 187-192) utilized the cDNA microarray approach to ascertain gene expression profiles of human coronary artery VEC treated with nicotine. Their analysis of over 4,000 genes identified a number of nicotine-modulated genes involved in signal transduction and transcriptional regulation. Changes in gene expression profiles associated with endothelial senescence have been investigated using cDNA array hybridization with mRNA isolated from late vs. early passages of dermal VEC (Vasile et al., 2001, FASEB 15: 458-466). The study results suggest that the expression of thymosin beta-10, a G-sequestering peptide involved in actin regulation, was strongly down regulated in senescent endothelial cells.
Despite this prior art, patterns of gene expression in tumor derived VEC remain poorly described. A group of hypoxia-induced genes that included TPA-1, insulin-like growth factor-binding receptor, endothelin-2, low-density lipoprotein-like receptor-related protein and some other markers of endothelial cells, were identified using cDNA microarrays hybridized with mRNA from two squamous cell carcinoma-derived tumor cell lines (Koong et al., 2000, Cancer Res. 60: 883-887). Joki et al. (2001,Neurosurgery 48: 195-201) utilized microarray technology to evaluate the effects of radiotherapy on gene expression in glioblastoma multiforme, and in particular, expression of those genes the products of which might influence the biology of neighboring tumor VEC. This reference disclosed decreased expression of growth factors participating in paracrine loops, such as VEGF and platelet-derived growth factor (PDGF) receptor beta, in four post-radiation recurrent tumors and correlated these changes with decreased microvessel counts in these tumors. A 332-membered human cDNA array was used to assess tumorigenesis- and angiogenesis-related patterns of gene expression in five normal ovary and four poorly differentiated serous papillary ovarian adenocarcinoma samples (Martoglio et al., 2000, Mol. Med. 6: 750-765). The transcription profiles analysis revealed an overall increase in angiogenesis-related markers, such as VEGF and angiopoietin-1 in tumor specimens. These changes were accompanied by the up-regulation of apoptotic/neoplastic markers (e.g., BAD, b-myb), immune response mediators (e.g., HLA-DR), and ovarian-specific biomarkers (e.g., cofilin, moesin, etc.). However, direct analysis of VEC that were physically isolated from tumor samples was not performed in these studies.
The most comprehensive large-scale analysis of gene expression in tumor-derived VEC was performed by St. Croix et al., who utilized SAGE libraries of approximately 193,000 14-base pair tags derived from a specific position near the 3′ termini of individual mRNA transcripts and corresponding to 32,500 unique transcripts (St. Croix et al, 2000, Science 289: 1197-1202). VEC were purified from dissociated human colorectal tumors using a two-step immunomagnetic beads base selection protocol including (i) negative selection of epithelial and hematopoietic cell populations on the basis of membrane antigenic markers, and (ii) positive selection of VEC based on the membrane binding of endothelial-specific P1H12 monoclonal antibody (mAb). The expression of candidate transcripts was confirmed by IHC and RT-PCR analyses. The authors reported a series of tags corresponding to either known or unknown genes that provided a first definitive molecular characterization of VEC derived from colorectal tumors. The top 25 tags with the highest tumor EC to normal EC ratios included several MMPs, collagens types I and III, enactin, cystatin S, endo 180 lectin, as well as several expression sequence tags (EST's) corresponding to yet unknown genes. Although gene expression was examined by St. Croix et al. in highly purified tumor VEC populations, no cDNA microarray experiments were performed in this study.
Gene array technologies have not been successfully applied to the analysis of tumor-derived VEC. Several issues have complicated cDNA microarray-based gene expression studies of endothelial cells in vitro. Obtaining pure cell populations from tumor biopsy specimens has been difficult because of the diverse mixture of cells that make up tumors, such as malignant, stromal and blood components (Emmert-Buck et al., 2000, J. Mol. Diagn. 2: 60-66). Additionally, VEC represent only a small fraction of the cells comprising the tumor sample. As a result, extracted RNA has been representative of a mixture of the cellular subsets, making it difficult to attribute specific gene expression patterns to the malignant component. There have been unsuccessful attempts in the art to purify target cells including laser capture microdissection, selective protection of malignant cells from irradiation, magnetic beads and flow cytometry (Shibata, 1998, Methods Mol. Biol. 92: 39-47; Conrad-Lapostolle et al., 1996, Cell Biol. Toxicol. 12: 189-197; Rosfiord et al., 1999, Methods Mol. Biol. 129: 79-90; Diez et al., 1999, J. Biochem. Biophys. Methods 40: 69-80; Auerbach et al., 1994, Proc. Annu. Meet Am. Assoc. Cancer Res. 35: 663).
Thus, there is a need in this art for developing methods for obtaining pure VEC populations from tumor biopsy samples, and for identifying gene expression patterns of VEC that are either sensitive or resistant to anti-angiogenesis agents, in order to identify agents that will be effective against angiogenesis. There is also a need for methods that provide additional information to physicians and cancer patients to enable more informed and individualized treatment decisions, particularly information relating to the usefulness of treating a cancer patient with anti-angiogenesis agents, thereby informing both physician and patient about the treatment methods that have the greatest likelihood of producing a positive outcome.